Method for diagnosis, prognosis and determination of treatment for cutaneous t-cell lymphoma

ABSTRACT

The present invention relates to biomarkers associated with CTCL, including TOX, PLS3, KIR3DL2, GATA3 and RUNX3, where increased expression, relative to normal control, of one or more of TOX, PLS3, KIR3DL2, and/or GATA3 is associated with CTCL and decreased expression of RUNX3, relative to normal control, is associated with CTCL. One or more of these biomarkers may be used to diagnose CTCL and/or design and monitor treatment.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.16/581,959, filed Sep. 25, 2019, which is a division of U.S. patentapplication Ser. No. 14/819,950, filed Aug. 6, 2015, now U.S. Pat. No.10,473,661, issued Nov. 12, 2019, which is a continuation ofInternational Patent Application No. PCT/US2014/015314, filed Feb. 7,2014, which claims priority to U.S. Provisional Application No.61/762,167, filed Feb. 7, 2013, to each of which priority is claimed andthe contents of each of which are incorporated herein by reference intheir entireties.

GRANT INFORMATION

This invention was made with government support under Grants Nos.CA121973, UL1TR000005 and RR024153 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

1. INTRODUCTION

The present invention relates to biomarkers of cutaneous T-cell lymphomaand Sézary Syndrome which may be used to diagnose these conditions andguide therapeutic regimens.

2. BACKGROUND OF THE INVENTION

T-cell lymphoma that involves the skin is generally known as cutaneoust-cell lymphoma (CTCL). The term CTCL encompasses a number of disorders,including mycosis fungoides (MF), which is the most common form of CTCL.Sézary syndrome (SS) is an advanced, variant form of mycosis fungoides,characterized by the presence of malignant lymphocytes in the blood. See“Getting the Facts” monograph for “Cutaneous T Cell Lymphoma” publishedby the Lymphoma Research Foundation, 115 Broadway Suite 1301, New YorkN.Y. 10006 (last update January 2013). No specific diagnostic orprognostic markers exist to enable early diagnosis of MF, SS and CTCL.The pathogenesis of CTCL is unknown and there are no therapeutic targetsavailable for disease-specific therapies. Because no specific diagnosisis possible, CTCL is often confused with other inflammatory dermatoses,such as psoriasis or eczema, and patients often live with the diseasefor years before a correct diagnosis is established. If diagnosed early,CTCL patients have a longer lifespan, but the survival rate goes down asCTCL reaches advanced stages. Thus, a diagnostic test that could easilyconfirm the presence of CTCL early in disease course is needed.

3. SUMMARY OF THE INVENTION

The present invention relates to biomarkers associated with CTCL and/orMF or SS in particular, including TOX, PLS3, KIR3DL2, GATA3 and RUNX3,where increased expression, relative to normal control, of one or moreof TOX, PLS3, KIR3DL2, and/or GATA3 is associated with CTCL anddecreased expression of RUNX3, relative to normal control, is associatedwith CTCL. Detection of these and/or a subset of these biomarkers may beused to diagnose CTCL and/or design and monitor treatment.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Genes selected for further study, showing expression, relativeto norm, in Séary syndrome (SS) and mycosis fungoides (MF).

FIGS. 2A-2G. Relative expression of (FIG. 2A) PLS3, KIR3DL2 and TOX,(FIG. 2B) GATA3 and (FIG. 2C) RUNX3 in SS versus normal CD4+ cells.(FIG. 2D) Expression levels of RUNx3 and TOX were inversely related.(FIG. 2E) Results of qRT-PCT (dark bars) versus sequence-basedtranscriptome analysis (light bars) for TOX, KIR3DL2 and PLS3. (FIG. 2F)TOX expression in individual subjects. (FIG. 2G) PLS3 expression inindividual subjects.

FIG. 3. Three components of the TOX-RUNX3 pathway are dysregulated.

FIG. 4. Expression of TOX and PLS3 (T-Plastin) in psoriasis, MF and SS.

FIGS. 5A-5C. Following romidepsin treatment, expression of (FIG. 5A)TOX; (FIG. 5B) PLS3 and (FIG. 5C) RUNX3 genes were measured.

FIG. 6. Fold-change relative to untreated of TOX and RUNX3 geneexpression after treatment with TOX siRNA.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to biomarkers for CTCL and associateddisorders (for example SS and/or MF) which may also serve as therapeutictargets. In certain non-limiting embodiments, the present inventionprovides for a method of diagnosing CTCL or an associated disorder in asubject comprising determining the level of expression of one or more(or two or more or three or more or four or more or five or more or sixor more or seven) of TOX, PLS3, KIR3DL2, GATA3, ITGB1, PDCD6 and/orRUNX3 in a sample of cells of a subject, relative to gene expression ina normal healthy subject, where increased expression in one or more ofTOX, PLS3, KIR3DL2 and/or GATA3 indicates a diagnosis of CTCL and/ordecreased expression of RUNX3 indicates a diagnosis of CTCL. In certainnon-limiting embodiments, the biomarkers tested include at least RUNX3.In certain non-limiting embodiments, the biomarkers tested include atleast TOX. In certain non-limiting embodiments, the biomarkers testedinclude at least KIR3DL2. Where the marker is TOX or ITGB1, an increasedlevel which is increased less than five-fold relative to normalindicates a diagnosis of the CTCL ,MF. Where the marker is TOX, KIR3DL2,ITGB1 or PDCD6, an increased level which is increased more thanfive-fold or more than 10-fold relative to normal indicates a diagnosisof the CTCL, SS.

In certain non-limiting embodiments, the present invention provides fora method of diagnosing CTCL or an associated disorder in a subjectcomprising determining the level of expression of RUNX3 in a sample ofcells of a subject, relative to gene expression in a normal healthysubject, where decreased expression of RUNX3 indicates a diagnosis ofCTCL. Related embodiments, in addition to determining expression ofRUNX3, further comprise determining the level of expression of one ormore (or two or more or three or more or four or more or five or more orsix) of TOX, PLS3, KIR3DL2, ITGB1, PDCD6 and/or GATA3 in a sample ofcells of a subject, relative to gene expression in a normal healthysubject, where increased expression in one or more of TOX, PLS3,KIR3DL2, ITGB1, PDCD6 and/or GATA3 indicates a diagnosis of CTCL. Incertain non-limiting embodiments, the biomarkers tested include, inaddition to RUNX3, at least TOX. In certain non-limiting embodiments,the biomarkers tested include, in addition to RUNX3, at least KIR3DL2.In certain non-limiting embodiments, the biomarkers tested include, inaddition to RUNX3, at least PLS3. Where the marker is TOX or ITGB1, anincreased level which is increased less than five-fold relative tonormal indicates a diagnosis of the CTCL ,MF. Where the marker is TOX,KIR3DL2, ITGB1 or PDCD6, an increased level which is increased more thanfive-fold or more than 10-fold relative to normal indicates a diagnosisof the CTCL, SS. Where the marker is PLS3, an increased level which isincreased more than ten-fold relative to normal indicates a diagnosis ofCTCL and an increased level which is increased more than 50 foldindicates SS. The subject is a human subject. In non-limitingembodiments, the subject has a skin lesion. In non-limiting embodiments,the subject has leukemic cells, for example but not limited to in aperipheral blood sample. In non-limiting embodiments, the subject has askin lesion and leukemic cells.

Expression levels may be determined, for example, by determining, in asample of cells from a subject (e.g. a human subject), levels of RNA(for example, using PCR analysis (or other method which includesamplification of RNA in the sample), array analysis, Northern blotanalysis, or any method known in the art) or by determining levels ofprotein expression (for example using antibodies or related molecules(e.g. single chain antibodies, antibody fragments, etc) or any methodknown in the art). Non-limiting specific examples of methods ofmeasurement may comprise antibody-binding, sequencing, probehybridization, real-time quantitative reverse transcription polymerasechain reaction (“RT-q PCR”), real time PCR, Northern blot,immunohistochemistry, and/or Western blot.

In one specific non-limiting embodiment, TOX may have NCBI ReferenceSequence NP_055544.1, Accession No. NP_055544 XP_376776.

In one specific non-limiting embodiment, PLS3 may have NCBI ReferenceSequence NP_001129497.1, Accession No. NP_001129497 and/or see Lin etal., 1999, DNA Cell Biol. 18:27-37.

In one specific non-limiting embodiment, for KIR3DL2, see NCBI Gene IDNo. 3812.

-   -   In one specific non-limiting embodiment, GATA3 may have NCBI        Reference Sequence NP_001002295.1, Accession No. NP_001002295.    -   In one specific non-limiting embodiment, RUNX3 may have GeneBank        No. AAH13362.1, Accession No. AAH13362.    -   In one specific non-limiting embodiment, ITGB1 may have GenBank        No. AAI13902, Accession No. AAI13902.    -   In one specific non-limiting embodiment, PDCD6 may have GenBank        No. AAH50597.1, Accession No. AAH50597.

In certain non-limiting embodiments the sample of cells is obtained froma region of diseased tissue in the subject or may be obtained from ablood sample. For example, but not by way of limitation, the sample maybe peripheral blood mononuclear cells, or CD4+ T cells, or cells fromtissue affected by the disease such as cells from a skin lesion.

In related embodiments, the invention provides for kits comprising ameans of determining expression levels of one or more (or two or more orthree or more or four or more or five or more or six or more or seven)of TOX, PLS3, KIR3DL2, GATA3, ITGB1, PDCD6 and/or RUNX3, optionallytogether with a positive and/or negative control. Such means maycomprise, for example but not by way of limitation, an antibody orfragment thereof or single chain antibody specific for the biomarker orbiomarkers to be detected, and/or a nucleic acid probe or primerspecific for the biomarker or biomarkers tested; these may be directlydetectable themselves or indirectly detectable, for example using alabeled secondary antibody or probe or a substrate.

An indication of a diagnosis of a CTCL would be desirably considered inconjunction with clinical features of a subject's presentation toconfirm a diagnosis, for example the appearance, symptomatology andhistopathology of skin lesions or the presence of atypical lymphocytesin the blood. A positive result showing increased expression of one ormore of these genes may be followed by one or more further diagnosticmeasure, for example, tissue histopathologic analysis, evaluation ofPBMC to look for leukemic cells, and/or one or more therapeutic measureto treat CTCL.

The present invention is further based, at least in part, on thediscovery that the expression levels of these genes can change inresponse to various treatments. Accordingly, the present inventionprovides a method for determining an effective treatment regimen for asubject suffering from CTCL comprising obtaining a cell sample from thesubject, determining the expression level of one or more (or two or moreor three or more or four or more or five) of TOX, PLS3, KIR3DL2, GATA3,and/or RUNX3 in the cells, exposing cells in the sample to a candidateCTCL therapeutic agent, and determining the expression level of thegene(s) in treated cells, where a decrease in the expression of one ormore of TOX, PLS3, KIR3DL2 and/or GATA3 in response to treatment and/oran increase in expression of RUNX3, resulting from treatment, indicatesthat the agent would be a beneficial therapy for the subject; suchmethod may be followed by treating the subject with the agent.Similarly, the expression level of one or more of TOX, PLS3, KIR3DL2,GATA3 and/or RUNX3 may be monitored in cells of a subject during thecourse of CTCL therapy, where a decrease in TOX, PLS3, KIR3DL2 and/orGATA3 and/or an increase in RUNX3 following treatment would beindicative of therapeutic benefit. In a certain non-limiting embodiment,the invention provides for a method for determining an effectivetreatment regimen for a subject suffering from cutaneous T-celllymphoma, comprising determining the expression level of one or more ofTOX, PLS3, KIR3DL2, GATA3, and/or RUNX3 in a sample of cells from thesubject prior to treatment with a therapeutic agent, administering thetherapeutic agent to the subject, and then determining the expressionlevel of one or more of TOX, PLS3, KIR3DL2, GATA3, and/or RUNX3 in asample of cells from the subject during or following treatment with thetherapeutic agent, where a decrease in the expression of one or more ofTOX, PLS3, KIR3DL2 and/or GATA3 in response to treatment and/or anincrease in expression of RUNX3, resulting from treatment, indicatesthat the agent has therapeutic benefit. It may be desirable to attemptto treat a subject with a less-agressive form of therapy, wherebiomarkers indicate a therapeutic benefit. Further, an increase in oneor more of TOX, PLS3, KIR3DL2 and/or GATA3 expression and/or a decreasein RUNX3 could indicate a need for more aggressive therapy. For examplebut not by way of limitation a decrease of TOX, PLS3, KIR3DL2 and/orGATA3 associated with therapeutic benefit may be a decrease of at leastabout 30 percent or at least about 50 percent relative to pre-treatmentlevels. For example but not by way of limitation an increase in RUNX3indicative of therapeutic benefit may be an increase of at least about30 percent or at least about 50 percent relative to pre-treatment level.In certain non-limiting embodiments, the biomarkers tested include atleast RUNX3. In certain non-limiting embodiments, the biomarkers testedinclude at least TOX. In certain non-limiting embodiments, thebiomarkers tested include at least PLS3. In certain non-limitingembodiments, the biomarkers tested include at least KIR3DL2. In certainnon-limiting embodiments, the biomarkers tested include at least TOX andRUNX3. In certain non-limiting embodiments, the biomarkers testedinclude at least PLS3 and RUNX3.

In particular non-limiting embodiments, the therapeutic agent may becorticosteroid, retinoid, imiquimod, radiation, methotrexate, UV light,romidepsin (e.g.,) Istodax®), photophoresis, noscapine (e.g., Targetin)or a noscapine analog, pralatrexate (e.g.,) Folotyn®, bortezomib(e.g.,)Velcade®, denileukin diftitox (e.g., Ontak®), vorinostat (e.g.,)Zolinza®, mechlorethamine gel (e.g., Valchlor™), alemtuzumab (e.g.,Campath®), liposomal doxorubicin, gemcitabine (e.g., Gemzar®),everolimus (e.g., Afinitor®), lenalidomide (e.g., Revlimid®),brentuximab vedotin (Adcetris®), panobinostat, forodesine, AP0866(a.k.a. Daporinad), mogamulizumab (KWO761), or a combination thereof Inparticular non-limiting embodiments, the therapeutic agent is a histonedeacetylase inhibitor. Non-limiting examples of “less-aggressive”therapeutic agents include corticosteroid, retinoid, imiquimod,radiation, methotrexate, and UV light phototherapy and photophoresis;non-limiting examples of more aggressive therapeutic agents includenoscapine (e.g., Targetin) or a noscapine analog, pralatrexate (e.g.,Folotyn®), bortezomib (e.g., Velcade®), denileukin diftitox (e.g.,Ontak®), vorinostat (e.g., Zolinza®), and mechlorethamine gel (e.g.,Valchlor); and non-limiting examples of even more aggressive agentsinclude alemtuzumab (e.g., Campath®), liposomal doxorubicin, andgemcitabine (e.g., Gemzar®).

See also Lessin et al., 2013, JAMA Dermatol. 149(1):25-32; Weberschocket al., 2012, Cochrane Database Syst. Rev. September 12; 9:CD008946; andKim et al., 2003, Arch. Dermatol. 139(7):857-866.

In still further embodiments, the present invention provides a method oftreating

CTCL comprising administering, to a subject in need of such treatment,an agent that reduces expression of TOX, where such agent is siRNA,antisense RNA, or a catalytic DNA or RNA which degrades TOX mRNA.

6. EXAMPLE 1: COMPONENTS OF THE TOX-RUNX3 PATHWAY ARE DIFFERENTIALLYEXPRESSED IN CUTANEOUS T-CELL LYMPHOMA

A sequence-based transcriptome approach that focuses on malignant cellsin Séary Syndrome (SS) and mycosis fungoides (MF) was used to identifygenes that are dysregulated in CTCL and which may serve as biomarkers ortargets for treatment. Five genes were selected for further study,namely T-plastin (PLS3), Thymocyte selection-associated high mobilitygroup box protein (TOX), Killer cell immunoglobulin-like receptor 3DL2(KIR3DL2), Integrin β1 (ITGB1) and Programmed cell death 6 (PDCD6) (FIG.1).

Quantitative real-time polymerase chain reaction (qRT-PCR) was used tomeasure expression of genes in CD4+ T cells from SS patients versusnormal controls. Table 1 shows the profiles of the SS patients studied.

TABLE 1 Age/Sex/ Patient Race Diagnosis, Stage Current Tx 1 58 WMT3N1M0B2/Stage IVA1, SS Romidepsin 2 80 WF T3N0M0B2/Stage IVA1, SSPhotophoresis, Targretin 3 42 AAF T3N1M0B2/Stage IVA1, SS Romidepsin 467 WF T3N0M0B2/Stage IVA1, SS Photophoresis 5 67 WM T3N0M0B2/Stage IVA1,SS Romidepsin 6 86 WF T3N0M0B2/Stage IIB, MF Pralatrexate, TargretinAs shown in FIG. 2A-B, expression of PLS3, KIR3DL2, TOX and GATA3 wereall relatively increased in the CD4+ cells of SS patients, whereasexpression of RUNX3 was relatively decreased (FIG. 2C). Expressionlevels of RUNX3 and TOX were inversely related (FIG. 2D). Results ofqRT-PCT versus sequence-based transcriptome analysis for TOX, KIR3DL2and PLS3 are shown in FIG. 2E. TOX expression in individual subjects isshown in FIG. 2F and PLS3 expression in individual subjects is shown inFIG. 2G.

TOX is a transcription factor with known downstream effects (Collins Aet al. Nat Rev Imm. 9: 106-115. 2009) and RUNX3 is a tumor suppressor(Chuang LSH et al. Oncogene. 29: 2605-2615. 2010). The data shown inFIG. 2A-C indicate that in CD4+ cells of SS patients, three componentsof the TOX-RUNX3 pathway are dysregulated (FIG. 3).

TOX and PLS3 may be used as diagnostic markers to distinguish psoriasis,SS and MF where expression is substantially elevated in SS and MFrelative to expression in psoriasis (FIG. 4); TOX and PLS3 may also beused as markers in diagnosis of CTCL.

7. EXAMPLE 2

Isolated PBMCs were cultured overnight and the next day divided into twotreatment groups: 4 μM romidepsin and 20 uM pralatrexate. Each treatmentwas added 24 hours following culture initiation and was replaced alongwith fresh media 24 hours after initial treatment. After 48 hours ofincubation with treatment, RNA was isolated from treated cells usingRNAprotect and RNeasy (Qiagen, Valencia, Calif.) per manufacturer'sinstructions. For cDNA synthesis, total RNA (100 ng) was used forReverse Transcription (RT) with Superscript II reverse transcriptase(Life Technologies, Gaithersburg, Md.) using oligo dT primers accordingto the recommendations of the manufacturer. 2 ul of the resulting cDNAwas used for each PCR reaction. Quantitative reverse transcription PCRwas performed using TaqMan PCR master mix (Applied Biosystems, FosterCity, Calif.) together with TaqMan probes and primers (AppliedBiosystems) using standard conditions. Expression of TOX, PLS3 and RNX3genes were measured in this way.

Treatment with romidepsin decreased gene expression of both TOX and PLS3(p<0.05) (FIG. 5A-B), and increased expression of RUNX3 (p<0.05)(FIGURE5C) resulting in normalization of gene expression. Treatment withpralatrexate did not result in altered gene expression of any genetested.

8. EXAMPLE 3

5 ng of TOX Silencer Select Validated siRNA (Ambion, Carlsbad, Calif.)was added along with 1.5 μL of TransIT-siQUEST Transfection Reagent(Minis, Madison, Wis.) per manufacturer's instruction to cultured PBMCsfrom patients 24 hours after culture was initiated. 72 hours later, RNAwas isolated from treated cells using RNAprotect and RNeasy (Qiagen,Valencia, Calif.) per manufacturer's instructions. For cDNA synthesis,total RNA (100 ng) was used for Reverse Transcription (RT) withSuperscript II reverse transcriptase (Life Technologies, Gaithersburg,Md.) using oligo dT primers according to the recommendations of themanufacturer. 2 μl of the resulting cDNA was used for each PCR reaction.Quantitative reverse transcription PCR was performed using TaqMan PCRmaster mix (Applied Biosystems, Foster City, Calif.) together withTaqMan probes and primers (Applied Biosystems) using standardconditions.

As shown in FIG. 6, siRNA knockdown of TOX resulted in decreased TOXexpression and increased RUNX3 expression. siRNA knockdown of PLS3 alsowas found to decrease PLS3 expression.

9. REFERENCES

1. Booken, N., A. Gratchev, et al. (2008). “Sezary syndrome is a uniquecutaneous T-cell lymphoma as identified by an expanded gene signatureincluding diagnostic marker molecules CDO1 and DNM3.” Leukemia 22:393-399.

2. van Doom, R., R. Dijkman, et al. (2004). “Aberrant Expression of theTyrosine Kinase Receptor EphA4 and the Transcription Factor Twist inSezary Syndrome Identified by Gene Expression Analysis ” Cancer Res 64:5578-5586.

3. Nebozhyn, M., A. Loboda, et al. (2006). “Quatitative PCR on 5 genesreliably identifies CTCL patients with 5% to 99% circulating tumor cellswith 90% accuracy.” Blood 107: 3189-3196.

4. Zhang, Y., Y. Wang, et al. (2012). “Molecular Markers of Early-StageMycosis Fungoides.” J Invest Dermatol 132: 1698-1706. 5. US 20060271309A1, Method of Diagnosis of Cancer Based on Gene Expression Profiles inCells, Showe et al.

6. US 20120288499 A1, Methods for diagnosis and treatment of Cutaneous Tcell lymphomas, Bensussan et al.7. US 20080274468 A1, Novel means for diagnosis and therapy of CTCL,Bensussan et al.8. US 20100035971 A1, Methods and means related to diseases, Ranki etal.9. Dulmage and Geskin (2013) “Lessons learned from gene expressionprofiling of cutaneous T-cell lymphoma.” Br. J. Dermatol. 169:1188-1197.

10. Lessin et al., 2013, JAMA Dermatol. 149(1):25-32.

11. Weberschock et al., 2012, Cochrane Database Syst. Rev. September 12;9:CD008946.12. Kim et al., 2003, Arch. Dermatol. 139(7):857-866.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

What is claimed is:
 1. A method of diagnosing cutaneous T-cell lymphoma(CTCL) in a subject, comprising: (a) obtaining a biological sample froma subject; (b) contacting the biological sample with means fordetermining expression levels of TOX, PLS3, KIR3DL2, GATA3, and RUNX3;(c) determining levels of expression of TOX, PLS3, KIR3DL2, GATA3, andRUNX3 in the biological sample of the subject relative to expressionlevels in a biological sample of a subject who does not suffer fromCTCL, (d) providing a diagnosis of CTCL, wherein the determinedexpression level of TOX is more than 5 fold; the determined expressionlevel of PLS3 is more than 10 fold; the determined expression level ofKIR3DL2 is more than 5 fold; the determined expression level of GATA3 ismore than about 1.75 fold; and the determined expression level of RUNX3is less than about 60%; all relative to the expression levels of asubject who does not suffer from cutaneous T-cell lymphoma.
 2. Themethod of claim 1, where expression is determined using quantitativereal-time polymerase chain reaction or antibody-mediated detectionmethods.
 3. The method of claim 1, where expression is determined by amethod comprising one or more of determining levels of RNA, arrayanalysis, Northern blot analysis, determining levels of proteinexpression, antibody-binding, probe hybridization, real-timequantitative reverse transcription polymerase chain reaction (RT-qPCR),real time PCR, immunohistochemistry, and/or Western blot.
 4. The methodof claim 1, wherein the expression levels of TOX, PLS3, KIR3DL2, GATA3,and RUNX3 are determined by a polymerase chain reaction (PCR) and/or areal-time PCR.
 5. The method of claim 1, wherein the expression levelsof TOX, PLS3, KIR3DL2, GATA3, and RUNX3 are determined by a quantitativereal-time PCR and/or a quantitative reverse transcription PCR.
 6. Themethod of claim 1, wherein the expression levels of TOX, PLS3, KIR3DL2,GATA3, and RUNX3 are determined by immunohistochemistry.
 7. The methodof claim 1, wherein the biological sample obtained from the subject is ablood sample.
 8. The method of claim 1, wherein the biological sampleobtained from the subject comprises peripheral blood mononuclear cellsobtained from the subject.
 9. The method of claim 1, wherein the sampleobtained from the subject comprises peripheral blood CD4+ T-cells. 10.The method of claim 1, wherein the subject has a skin lesion.
 11. Themethod of claim 1, wherein the sample obtained from the subjectcomprises cells of the skin lesion.